Determination of an analyte in a liquid medium

ABSTRACT

The present invention concerns a magneto-controlled method and system for the determination of an analyte in a liquid medium. The method and system of the invention are based on the use of functionalized magnetic particles, e.g. magnetic particles that carry a recognition agent, such that in the presence of the analyte and under appropriate conditions, a chemical reaction occurs yielding a reaction signal. The reaction signal may be an electric signal, a colorimetric signal, light emission or the formation of a precipitate. In accordance with the invention the reaction is significantly enhanced by inducing rapid vibrations or rotations of the magnetic particles on the barrier surface.

FIELD OF THE INVENTION

This invention relates to a method for detecting an analyte in anassayed sample. More specifically, the present invention concerns amagneto-controlled method for determination of an analyte in a liquidmedium.

LIST OF REFERENCES

The following references are considered to be pertinent for the purposeof understanding the background of the present invention:

-   1. Hirsch, R.; Katz, E.; Williner, I.; J. Am. Chem. Soc. 2000, 122,    12053-12054.-   2. Katz, E.; Sheeney-Haj-Ichia, L.; Wiliner, I., Chem. Eur. J. 2002,    8, 4138-4148.-   3. Katz, E.; Sheeney-Haj-Ichia, L.; Buckmann, A. F.; WilIner, I.;    Angew. Chem. Int. Ed. 2002, 41, 1343-1346.-   4. Sheeney-Haj-Ichia, L.; Katz, E.; Wasserman, J.; Willner, I.;    Chem. Commun. 2002, 158-159.-   5. Katz, E.; Willner, I.; Electrochem. Commun. 2002, 4,201-204.-   6. Dickson, D. P. E.; Walton, S. A.; Mann, S.; Wong, K.; NanoStruct.    Mater. 1997, 9, 595-598.-   7. De Cuyper, M.; Joniau, M.; Biotechnol. Appl. Biochem. 1992, 16,    201-210.-   8. Carpenter, E. E.; J. Magnetism Magnetic Mater. 2001, 225, 17-20.-   9. Matsunaga, T.; Takeyama, H.; Supramolec. Sci. 1998, 5, 391-394.-   10. Liao, M.-H.; Chen, D.-H.; Biotechnol. Lett. 2001, 23, 1723-1727.-   11. Mornet, S.; Vekris, A.; Bonnet, J.; Duguet, E.; Grasset, F.;    Choy, J.-H.; Portier, J.; Mater. Lett. 2000, 42, 183-188.-   12. Sonti, S. V.; Bose, A.; J. Colloid Interface Sci. 1995, 170,    575-585.-   13. Shen, L.; Laibinis, P. E.; Hatton, T. A.; Langmuir 1999, 15,    447453.-   14. Katz, E.; Lotzbeyer, T.; Schlereth, D. D.; Schuhmann, W.;    Schmidt, H.-L.; J. Electroanal. Chem. 1994, 373,189-200.-   15. Bard, A. J.; Faulkner, L. R.; Electrochemical Methods:    Fundamentals and Applications, Wiley, New York, 1980.-   16. Moiroux, J.; Elving, P. J.; J. Am. Chem. Soc. 1980, 102,    6533-6538.-   17. Gorton, L.; J. Chem. Soc., Faraday Trans. 1, 1986, 82,    1245-1258.

The above publications will be referenced bellow by indicating theirnumber from the above list.

BACKGROUND OF THE INVENTION

Recent efforts are directed to the magnetic-field switching ofelectrocatalytic and bioelectrocatalytic processes.^(1,2) Severalapplications of magneto-controlled electron transfer reactions, such asselective dual biosensing,³ stimulated electrogeneratedchemiluminescence⁴ and selective patterning,⁵ were suggested. Magneticparticles functionalized with chemical or biological components areextensively used as a “collection tool” for the concentration and thelocalization of chemical or biochemical components.⁶⁻⁹ Differentapplications of magnetically-confined chemical components were reported,including transport and concentration of enzymes,¹⁰ DNA¹¹ or cells.¹²

SUMMARY OF THE INVENTION

The present invention provides a method and system for the determinationof an analyte in an assayed, liquid sample. The method and system of theinvention are based on the use of functionalized magnetic particles,e.g. magnetic particles that carry a recognition agent, such that in thepresence of the analyte and under appropriate assay conditions, areaction occurs yielding a reaction signal.

The term “reaction” is used to denote one or more reactions orinteractions carried out at once or in sequence, to yield the reactionsignal. The “reaction signal” is any detectable parameter that isyielded by the reaction. Accordingly, the term “assay conditions”encompasses all the conditions, substances or actions necessary oruseful for the appropriate reaction to take place, including sequencesof varying conditions or actions.

The particles are drawn to a barrier surface in the reaction cell,through a magnet placed in proximity to the barrier surface. Thereaction is detected by a sensing member, which forms the barriersurface or is part of the barrier surface, or is located in proximity tothe barrier surface or elsewhere.

The sensing member may be an electrode of an electrochemical cell andthe reaction signal in such example is an electric response that resultsfrom a reaction occurring as a result of the presence of an analyte inthe assayed sample. The term “electric response” refers to anymeasurable change in the electrical parameters recorded by or electricalproperties of the electrode. An electric response may be flow ofcurrent, charge or potential change, that results from a reactionoccurring at the surface of the electrode; a change in the amperometricresponse of the electrode that can be measured, for example, by means ofa cyclical voltamogram; etc. As will no doubt be appreciated, theinvention is not limited by the manner in which the electric response ismeasured and any manner of measurement that may be used therefor couldbe applied for measurement of the electric response in the method andsystem of the invention.

In addition to an electric response, other examples for the reactionsignal are the emission of light, a colorimetric response or theformation of a precipitate on the sensing member. Such responses may bemeasured by appropriate optical sensing means. The formation of aprecipitate on the sensing member may also be determined throughmeasuring of a change in the electric response of the sensing member,being in such case an electrode, for example using Faradaic impedancespectroscopy.

In accordance with the invention the reaction may be significantlyenhanced by inducing rapid movements, i.e. rapid vibrations or rotationsof the magnetic particles on the barrier surface. This may be achieved,for example, by a rotating motor associated with the magnet and thatcauses the magnet to rotate, and hence induces rotation of the magneticparticles.

The electrocatalytic and bioelectrocatalytic transformations at theparticles' interface are controlled, among others, by the rate oftransport of the analyte or of other substances that participate in theassay, towards the reaction site. Without wishing to be bound by theory,it is believed that the rotation or vibration of the magnetic particlesyields a hydrodynamic mass-transport of the analyte and/or assaysubstances towards the reaction site to facilitate the reaction betweenthe analyte and/or assay substances and the functionalized magneticparticles or any moiety attached thereto. Rotating or vibrating themagnetic particles through a rotating or vibrating magnetic field, is apreferred embodiment of the invention.

The invention permits the qualitative detection of the presence of ananalyte in an assayed sample by monitoring the occurrence of a reactionsignal. By measuring the extent of the signal, the concentration of theanalyte in the assay sample may also be quantitatively determined. Inthe following, the term “determination” or “determining” or “detection”will be used to refer collectively to both qualitative and quantitativeassay of the analyte in the assayed sample.

The term “magnet” will be used to denote both a passive magnet made of amagnetized metal alloy and an electromagnet.

According to one aspect of the invention, there is provided a method fordetermining an analyte in an assayed sample, comprising:

-   -   (i) providing magnetic particles carrying a recognition agent        that binds to or reacts with the analyte, such that, under assay        conditions, said binding or reaction yields a reaction signal;    -   (ii) contacting said magnetic particles with the assayed sample,        drawing the magnetic particles to a barrier surface through a        magnet proximal to the barrier surface, providing the assay        conditions and inducing the magnetic particles to rapidly rotate        or vibrate, giving rise to a reaction signal; and    -   (iii) reading said reaction signal.

The magnetic particles used in the method of the invention are typicallymade of Fe₃O₄, Fe, Co, Ni, their alloys, as well as other ferromagneticmaterials.

According to another aspect, the present invention provides a system fordetermining an analyte in an assayed sample, the system comprising:

-   -   (a) a cell with a barrier surface;    -   (b) a sub-system for causing the magnetic particles to rotate or        vibrate;    -   (c) magnetic particles having immobilized thereon a recognition        agent such that in the presence of the analyte, a reaction        occurs yielding a reaction signal, said signal being enhanced        during the rotation or vibration of said magnet;    -   (d) sensing member for sensing said reaction signal; and    -   (e) reader for reading said reaction signal.

Said sub-system, according to one embodiment of the invention, comprisesa motor associated with the magnet that causes the magnet to rapidlyrotate or vibrate.

The magnetic particles used in the system of the invention are typicallymade of Fe₃O₄, Fe, Co, Ni, their alloys, as well as other ferromagneticmaterials.

According to one embodiment of the invention the system is anelectrochemical system and the reaction that yields said reaction signalis a redox reaction.

The present invention is not limited by the nature of the recognitionagent and the analyte, the nature of the reaction that yields thereaction signal, the assay conditions or by the reaction signal. Thereare many types of reactions that permit detection of an analyte in amedium through immobilized recognition agents, such as those disclosedin WO 97/45720 and WO 00/32813 the contents of which are incorporatedherein by reference.

In accordance with one embodiment of the invention, the assayed sampleis first reacted to cause binding of the analyte, if present in thesample, with a recognition agent which may be a fluorescent or anothercalorimetric marker, a radio label, an enzyme that can catalyze adetectable reaction or a reagent that can undergo a redox reaction.

Accordingly, the recognition agent and the analyte can react with oneanother in a manner to yield a reaction product. The reaction istypically, but not exclusively, a redox reaction. The assay conditions,in accordance with this embodiment, comprise temperature conditions andreagents that permit the reaction to occur. The reagents that permit thereaction between the recognition agent and the analyte typically includea catalyst, for example, an enzyme that can catalyze this reaction.Specific examples of analytes that can be detected in accordance withthis embodiment include sugar molecules such as glucose, fructose,mannose, etc.; hydroxy or carboxy compounds, e.g. lactate, ethanol,methanol, formic acid, etc.; or amino acids. The recognition agents insuch cases are quinones, e.g. naphthoquinones, pyrroloquinoline quinone(PQQ), etc. An enzyme that can induce a reaction, in this case a redoxreaction, includes glucose oxidase, lactate dehydrogenase, fructosedehydrogenase, alcohol dehydrogenase cholin oxidase and the like.

In accordance with another embodiment, the recognition agent comprises acatalyst that can induce a reaction in which the analyte is convertedinto a product. In accordance with this specific embodiment, thereaction may be a redox reaction and the reaction may be monitoredthrough measuring the electric response of an electrode. Where thecatalyst is an enzyme, the identity of the enzyme determines specificityof the reaction.

Alternatively, the analyte may be a catalyst that can induce a reactionin which the recognition agent is converted into a product. Accordingly,the reaction signal would be such that is present only if therecognition agent was converted by the catalyst.

In accordance with yet another embodiment of the invention, the analyteand the recognition agent form a recognition pair. Examples ofrecognition pairs may be: antigen-antibody, ligand-receptor,oligonucleotide-oligonucleotide with a complementary sequence,oligonucleotide-binding protein, and sugar-lectin. The analyte is thenone of the pair and the detection moiety the other. The detection may bebased on the use of a reagent that binds to the formed couple, such asan agent that binds specifically to a double-stranded oligonucleotideand not to a single-stranded oligonucleotide, or an enzyme that usesonly double-stranded oligonucleotides and not single-strandedoligonucleotides as substrates.

In the alternative, detection may be based on a reagent that bindsspecifically to the analyte. In the latter case, the binding between theanalyte and the reagent is permitted first to occur and thereafter,excess reagents are removed and the reaction is allowed to proceed. Thereagent may be contacted with the analyte before, during or after therecognition agent is introduced. Examples of such reagents are anantibody or a nucleotide chain, capable of specific binding to theanalyte when it is bound to the recognition agent. The reagent may carrya detectable label, which may be a fluorescent, colorimetric or redoxlabel, or may be an agent that can by itself undergo a reaction orcatalyze a reaction such as an enzyme, an agent that can undergo a redoxreaction, etc.

In the method of the invention, during the analysis, at least one of thecomponents of the chemical system, for example the analyte, therecognition moiety or the catalyst, should be dissolved in the analyzedliquid medium, whereas the remaining component should be linked to themagnetic particles.

In accordance with yet another embodiment of the invention, the assaycomprises a first reagent capable of modifying the analyte, or a complexcomprising the analyte, such that the reaction product is detectable bya second reagent or more, ultimately yielding a reaction signal that isdependant on the presence or concentration of the analyte in the sample.One example of such assay is use of an enzyme to modify the recognitionagent in the presence of the analyte by binding a biotin-containingmoiety to the recognition agent. The biotin moiety bound to therecognition agent then serves as a specific binding site to a secondreagent comprising for example avidin-horseradish peroxidase (HRP) thatacts as a biocatalytic label. It is appreciated that this assay can leadalso to amplification of the signal, by repeatedly labeling more thanone molecule of the recognition moiety, such as using the polymerasechain reaction to label a recognition agent being single-stranded DNA inthe presence of a DNA analyte.

In accordance with another embodiment of the invention a method isprovided for the detection of cancer cells comprising:

-   -   (i) providing magnetic particles carrying a DNA recognition        agent that serves as a primer for telomerase, such that, under        assay conditions, the telomerase reaction enables a reaction        that yields a reaction signal;    -   (ii) providing an assay sample comprising cellular extract from        one or more cells suspected of being cancerous;    -   (iii) contacting said magnetic particles with the assayed        sample, drawing the magnetic particles to a barrier surface        through a magnet proximal to the barrier surface, providing the        assay conditions and inducing the magnetic particles to rapidly        rotate or vibrate, giving rise to a reaction signal;    -   (iv) reading said reaction signal; and    -   (v) comparing said reading with a reading obtained from a        control assay sample not containing cancerous cells, a higher        reading in the assay sample than in the control assay sample        indicating that said suspected cells are cancerous.

It is appreciated that according to this embodiment of the invention,cancer can be detected in tissue taken from a patient, in order todiagnose the patient's condition. Alternatively such tissue samples canbe taken during treatment of a known cancer patient in order to evaluatethe success or progress of the treatment. The term ‘cancer’ or‘cancerous’ are used to denote any cancerous or malignant condition of acell or a patient, whether human or not.

In the method of the invention, the presence of the analyte in themedium results in the formation of a signal, e.g. electrical signal,color signal, light emission or formation of a precipitate, therebyindicating the presence of the analyte. The sensing member is such thatcan sense the reaction signal. When the signal is emission of light thedetector is a light detector.

When the signal is electrical, it results from the transfer of electronsbetween an electrode and an electron transfer chain, where the analyteis a member of that electron transfer chain.

Electrodes suitable for use in the method of the invention are made ofor coated with conducting or semi-conducting materials, for examplegold, platinum, palladium, silver, carbon, copper, indium tin oxide(ITO), etc.

It would be appreciated that the methods and systems of the inventionare applicable also to the simultaneous or sequential detection of morethan one analyte. In such case, the magnetic particles would carry morethan one recognition agent (either on the same magnetic particle or ondifferent magnetic particles). In order for simultaneous detection totake place, the assay conditions should be such that would allow thesimultaneous formation of reaction signals that are distinguishable foreach analyte. Accordingly, the presence of one analyte would lead to areaction signal of one type (e.g. light emission) while the presence ofanother analyte would lead to a reaction signal of another type (e.g.formation of a precipitate on a sensing member, or emission of light ina different spectrum). Alternatively, the detection of the more than oneanalytes may be achieved in sequence, such that after one assay isperformed, the magnetic particles are collected, washed and providedwith different assay conditions for the detection of another analyte. Insuch case, the reaction signal may be the same, provided that in eachassay the reaction signal would be obtained solely in connection withthe presence of a single analyte.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to understand the invention and to see how it may be carriedout in practice, several preferred embodiments will now be described, byway of non-limiting examples and with reference to the accompanyingdrawings, in which:

FIG. 1 illustrates the functionalization of the magnetic particles withpyrroloquinoline quinone (PQQ) (1) or withN-(ferrocenylmethyl)aminohexanoic acid (2).

FIG. 2A shows cyclic voltammograms of an Au-electrode with themagnetically attracted PQQ-functionalized magnetic particles (10 mg) inthe presence of 50 mM NADH upon rotation of the magnet: (a) 0 rpm, (b)10 rpm, (c) 100 rpm, (d) 1000 rpm. Potential scan rate, 5 mV·s⁻¹.

FIG. 2B shows calibration plots for the amperometric detection of NADH(E=0.1 V) upon rotation of the magnet: (a) 0 rpm, (b) 100 rpm, (c) 1000rpm. The data were recorded in 0.1 M Tris-buffer, pH 7.0 with 20 mMCaCl₂.

FIG. 3A shows cyclic voltammograms of a Au-electrode with themagnetically attracted (2)-functionalized magnetic particles (6 mg) inthe presence of glucose oxidase, 1×10⁻⁵ M, and glucose, 50 mM uponrotation of the magnet: (a) 0 rpm, (b) 10 rpm, (c) 100 rpm, (d) 400 rpm.Potential scan rate, 5 mV·s⁻¹.

FIG. 3B shows calibration plots for the amperometric detection ofglucose (E=0.5 V) upon rotation of the magnet: (a) 0 rpm, (b) 100 rpm,(c) 400 rpm. The data were recorded in 0.1 M phosphate buffer, pH 7.0.

FIG. 4A illustrates an embodiment where the functionalization ofmagnetic particles is made with a DNA primer.

FIG. 4B illustrates a system implementing the embodiment illustrated inFIG. 4A, where the reaction signal is read by means of light emission.

FIG. 5 illustrates a system implementing an embodiment for thefunctionalization of the magnetic particles, similar to that illustratedin FIG. 4A but where the reaction signal is read by means of Faradaicimpedance.

FIG. 6A illustrates the functionalization of magnetic particles with anantigen.

FIG. 6B illustrates the functionalization of magnetic particles with anaphthoquinone (4).

FIG. 6C illustrates an immunosensing system implementing both theembodiments illustrated in FIGS. 6A and 6B.

FIG. 7A shows a plot of the intensity of the light emission vs. time,obtained in the system illustrated in FIG. 6C, without rotation of themagnetic particles (curve a) and with rotation (100 rpm, curve b).

FIG. 7B shows two calibration plots for the light signal intensity vs.the DNP-antibody concentration in the system illustrated in FIG. 6C:(a)—without rotation; and (b)˜with rotation (100 rpm).

FIG. 8A schematically illustrates the binding of a DNA analyte by use ofDNA-functionalized magnetic particles, biotin labeled DNA and anavidin-HRP conjugate.

FIG. 8B shows a detection system for a DNA analyte, implementing theDNA-functionalized magnetic particles illustrated in FIG. 8A togetherwith quinone-modified magnetic particles.

FIG. 9A shows a graph of chemiluminescence intensities upon the analysisof a DNA analyte (13), 1.4×10⁻⁸ M, according to FIGS. 8A and 8B, atdifferent rotation speeds: (a) 0 r.p.m.; (b) 60 r.p.m.; (c) 400 r.p.m.;(d) 2000 r.p.m.; (e) Analysis of mutant (13a), 1×10⁻⁷ M, at 2000 r.p.m.Inset: a graph showing the relation between the light intensity and ω²(ω=rotation speed). The chemiluminescence signals are produced byapplying a potential step on the electrode from 0 to −0.5 V and back(vs. SCE).

FIG. 9B shows a graph of light intensities as a function of theconcentration of a DNA analyte (13) according to FIGS. 8A and 8B, atvariable rotation speeds: (a) 0 r.p.m.; (b) 60 r.p.m.; (c) 2000 r.p.m.Inset: Enlargement of the results in the lower concentration range. Thechemiluminescence signals are produced by applying a potential step onthe electrode from 0 to −0.5 V and back (vs. SCE).

FIG. 10 shows amplified detection of viral DNA by multi-labeled rotatingmagnetic particles: (A) Labeling of the nucleic acid replica on theparticles with biotin units using thermal cycles. (B) Generation ofamplified chemiluminescence upon rotation of the functionalized magneticparticles on electrode surfaces.

FIG. 11 schematically shows the binding of a DNA primer as a recognitionagent to magnetic particles, using the heterobifunctional cross-linker3-maleimidopropionic acid N-hydroxysuccinimide ester.

FIG. 12 shows a graph of chemiluminescence intensities upon the analysisof M13φ DNA, 8×10⁻⁹M, at different rotation speeds, (a) 0 r.p.m.; (b) 60r.p.m.; (c) 400 r.p.m.; (d) 2000 r.p.m., and curve (e) chemiluminescencesignal upon applying the protocol in the absence of M13φ DNA at 2000r.p.m. Inset: Chemiluminescence intensities as a function of ω^(1/2)(ω=rotation speed). Chemiluminescence was generated by the applicationof a potential step from E=0.0V to E=−0.5V and back vs SCE. Arrows infigure indicate the times for switching the potential to −0.5V and to0.0V, respectively. Data recorded in 0.01M phosphate buffer pH=7.4 thatincludes luminol, 1×10⁻⁶M, under air.

FIG. 13 shows a calibration curve corresponding to the chemiluminescenceintensities upon analyzing different concentrations of M13 φ DNA at: (a)2000 r.p.m.; (b) 400 r.p.m.; (c) 60 r.p.m.; (d) 20 r.p.m.; (e) 0 r.p.m.

FIG. 14 shows the amplified detection of a single-base-mismatch in DNAusing magnetic particles

FIG. 15 depicts a graph of chemiluminescence intensities upon theanalysis of a DNA mutant sequence, (18), 1×10⁻⁹M at: (a) 2000 r.p.m.;(b) 400 r.p.m.; (c) 60 r.p.m. The chemiluminescence intensity upon theanalysis of the normal sequence, (19), 1.4×10⁻⁶M, at 2000 r.p.m. isshown in curve (d). The conditions for the recording of thechemiluminescence are detailed in FIG. 12.

FIG. 16 shows calibration curves corresponding to the analysis ofdifferent concentrations of the mutant (18) at different rotationspeeds: (a) 2000 r.p.m.; (b) 400 r.p.m.; (c) 60 r.p.m.; (d) 0 r.p.m.Inset: Enlargement of calibration curves showing the chemiluminescenceintensities at low concentrations of (18).

FIG. 17 schematically shows the amplified rapid detection of telomeraseactivity by multi-labeled rotating magnetic particles. (A)Multi-labeling of magnetic particles with biotin units as a result ofthe telomerase enzyme activity. (B) Generation of amplifiedchemiluminescence upon rotation of the biotin-multifunctionalizedmagnetic particles on electrode surfaces.

FIG. 18A shows chemiluminescence intensities upon the analysis of a293-kidney cancer cell extract containing 100,000 cells, at differentrotation speeds: 0 r.p.m.; 20 r.p.m.; 60 r.p.m.; 400 r.p.m.; 2000 r.p.m.Arrows indicate the times for switching the potential to −0.5 V and to0.0 V, respectively.

FIG. 18B shows chemiluminescence intensities as a function of ω^(1/2)(ω=rotation speed). In all experiments chemiluminescence was generatedby the application of a potential step from E1=0.0 V to E2=−0.5 V andback vs. SCE. Data recorded in 0.01 M phosphate buffer, pH=7.4, thatincludes luminol, 1×10⁻⁶M, under air.

FIG. 19 shows the calibration curves corresponding to thechemiluminescence intensities upon analyzing extracts of 293-kidneycancer cells of a different number of cells, at constant rotation speedsof (i) 0 r.p.m., (ii) 60 r.p.m. and (iii) 2000 r.p.m. Inset: Enlargementof calibration curves showing the chemiluminescence signal intensitiesobtained from extracts containing 0-100 cells. The conditions for therecording of the chemiluminescence are as detailed in FIG. 18.

FIG. 20A Shows chemiluminescence intensities upon the analysis of aHeLa-cells extract containing 100,000 cells, at: 0 r.p.m.; 20 r.p.m.; 60r.p.m.; 400 r.p.m.; 2000 r.p.m. Inset: Chemiluminescence intensities asa function of ω1/2 (ω=rotation speed). The conditions for the recordingof the chemiluminescence are detailed in FIGS. 18A, B.

FIG. 20B depicts the calibration curves corresponding to thechemiluminescence intensities upon analyzing extracts containing adifferent number of HeLa cells at constant rotation speeds of 0 r.p.m.,60 r.p.m. and 2000 r.p.m. Inset: Enlargement of calibration curvesshowing the chemiluminescence signal intensities obtained from extractscontaining: 0-100 cells.

FIG. 21 depicts electrogenerated chemiluminescence intensities obtainedfrom extracts containing: (a) 1000 HeLa cells; (b) 1000 293-kidneycancer cells; (c) 100,000 NHF cells.

FIG. 22 shows electrogenerated chemiluminescence intensity obtained uponanalyzing extracts from: (a) lung adenocarcinomas; (b) lung squamousepithelial carcinomas; (c) healthy tissues, (d) normal cells extract.

DETAILED DESCRIPTION OF THE INVENTION

It should be noted that during an analysis performed by the method ofthe invention, at least one of the components of the chemical system,for example the analyte, the recognition moiety, the catalyst or acomponent needed for the catalyst's activity such as a substrate, shouldbe dissolved in the analyzed liquid medium, whereas the other componentsare linked to the magnetic particles. In the examples below, thefollowing components were in the respective solutions:

-   -   (a) for the NADH analysis—NADH was dissolved in the solution and        PQQ was immobilized on the magnetic particles;    -   (b) for the analysis of glucose—glucose was dissolved in the        solution together with glucose oxidase that functions as a        biocatalyst, while ferrocene, which is the electron mediator        providing electrical communication between the electrode and the        enzyme, was immobilized at the magnetic particles;    -   (c) for the DNA analysis according to FIGS. 4A, 4B and        5—complementary DNA (the analyte) was immobilized on the DNA        functionalized magnetic particles together with doxorubicin        which functions as an electrocatalyst, while oxygen that is a        substrate electrocatalytically converted into hydrogen peroxide,        is soluble in the analyzed medium;    -   (d) antibody analysis—DNP-antibody is the analyte and was        immobilized at the particle surface together with an        electrocatalytic naphthoquinone.

Oxygen is the solubilized material that is convertedelectrocatalytically to hydrogen peroxide.

-   -   (e) for the DNA analysis according to FIGS. 8A, 8B and        9—complementary DNA (the analyte) was immobilized on the DNA        functionalized magnetic particles. An additional DNA reagent        complementary to the analyte was immobilized to said complex, to        which the enzyme horseradish peroxidase (HRP) was immobilized        via a biotin-avidin interaction. Naphthoquinone was also        immobilized to magnetic particles. Upon the application of a        potential on the electrode, the naphthoquinone is reduced to        hydroquinone and the electrocatalyzed reduction of oxygen to        hydrogen peroxide occurs. The HRP-catalyzed oxidation of luminol        by the electrogenerated hydrogen peroxide results in        chemiluminescence and emission of light. Luminol and hydrogen        peroxide are soluble in the analyzed medium;    -   (f) for the DNA analysis according to FIGS. 10-13—complementary        DNA (M13 φ DNA; the analyte) was immobilized on the DNA        functionalized magnetic particles and the complex was used as a        substrate for Taq-Polymerase. The nucleotides (DATP, dCTP, dTTP        and dGTP and biotin-dUTP) were soluble in the analyzed medium.        After the polymerase reaction has been terminated, HRP was        immobilized on the DNA linked magnetic particles via a        biotin-avidin interaction. Electrocatalytic naphthoquinone was        also immobilized to magnetic particles. Oxygen that is a        substrate electrocatalytically converted into hydrogen peroxide,        and luminol that, along with hydrogen peroxide is the substrate        for HRP is also soluble in the analyzed medium;    -   (g) for the DNA analysis according to FIGS. 14-16—complementary        DNAs (the mutant analyte and the wild-type DNA) were immobilized        on the DNA linked magnetic particles and the complex was used as        a substrate for Taq-Polymerase. The nucleotide, biotin-dCTP, was        soluble in the analyzed medium. After the polymerase reaction        has been terminated, HRP was immobilized on the DNA        functionalized magnetic particles via a biotin-avidin        interaction. Electrocatalytic naphthoquinone was also        immobilized to magnetic particles. Oxygen that is a substrate        electrocatalytically converted into hydrogen peroxide, and        luminol that, along with hydrogen peroxide is the substrate for        HRP is also soluble in the analyzed medium.

(h) For the telomerase analysis the enzyme analyte catalyzed theaddition of telomeric repeats to the DNA primer, which was bound to themagnetic particles. The nucleotides (DATP, dCTP, dTTP and dGTP andbiotin-dUTP) were soluble in the analyzed medium. After the telomerasereaction has been terminated, HRP was immobilized on the DNA linkedmagnetic particles via a biotin-avidin interaction. Electrocatalyticnaphthoquinone was also immobilized to separate magnetic particles.Oxygen that is a substrate, was electrocatalytically converted intohydrogen peroxide, and luminol that, along with hydrogen peroxide is thesubstrate for HRP is also soluble in the analyzed medium.

Magnetic particles (Fe₃O₄, ca. 1 μm average diameter, saturatedmagnetization ca. 65 emu·g⁻¹) were prepared according to the publishedprocedure¹³ without including the surfactant into the reaction medium.FIG. 1 illustrates the functionalization of the magnetic particles. Themagnetic particles were silanized with[3-(2-aminoethyl)aminopropyl]trimethoxysilane and then functionalizedwith pyrroloquinoline quinone, PQQ (1), or withN-(ferrocenylmethyl)aminohexanoic acid (2), using1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, EDC, as a couplingreagent. The PQQ-functionalized magnetic particles attracted to a bottomAu-electrode (0.24 cm²) by the external magnet (NdFeB/Zn-coated magnet,18 mm diameter, providing 0.2 kOe at the electrode surface), reveal areversible cyclic voltammogram at E°=−0.13 V (vs. SCE), pH=7.0,indicating an average surface coverage of 7500 PQQ units per particle.The cyclic voltammogram of the PQQ units associated with the magneticparticles is independent of the rotation-speed of the external magnet,indicating that the redox-units are confined to the electrode support.The PQQ-functionalized magnetic particles act as an electrocatalyst forthe oxidation of 1,4-dihydronicotineamide adenine dinucleotide, NADH,especially efficient in the presence of Ca²⁺ ions.¹⁴ FIG. 2A shows thecyclic voltammograms observed upon the PQQ-magnetite mediatedelectrocatalyzed oxidation of NADH, 50 mM, at different rotation-speedsof the external magnet. FIG. 2B shows the calibration curvescorresponding to the anodic currents originating in the presence ofdifferent concentrations of NADH at variable rotation speeds of theexternal magnet. From FIGS. 2A and 2B it is evident that the resultingelectrocatalytic currents increase as the rotation speed of the externalmagnet is elevated (the theoretical relation I_(cat) ∝ω^(1/2) isobserved at low rotation speeds).

In a control experiment, PQQ-functionalized silica particles thatgravimetrically settle on the Au-electrode, were subjected to differentrotation-speeds of the external magnet in the presence of NADH. Noeffect of the external rotating magnet on the resulting electrocatalyticcurrent was observed. This implies that the rotation of the magneticparticles on the electrode support leads to the increasedelectrocatalytic anodic currents upon rotation of the external magnetdue to hydrodynamic control of the substrate mass-transport to theelectrode.

The magnetic-field stimulated enhancement of the electrocatalyticcurrents generated by the rotation of redox-functionalized magneticparticles was also demonstrated for bioelectrocatalytic transformations.The magnetic particles functionalized with the ferrocene derivative,(2), were attracted to the Au-electrode and rotated on the conductingsupport by means of the external rotating magnet. The quasi-reversibleredox-wave of the ferrocene units, E°=0.32 V, is independent of therotation of the external magnet. FIG. 3A shows the cyclic voltammogramsof the ferrocene-functionalized magnetic particles in the presence ofglucose oxidase (GOx), 1×10⁻⁵ M, and glucose, 50 mM, at differentrotation rates of the external magnet. FIG. 3B shows the calibrationcurves corresponding to the amperometric responses of the system atdifferent concentrations of glucose and variable speeds of rotation ofthe external magnet. The electrocatalytic anodic currents increase asthe external rotation speed of the magnet is elevated. Controlexperiments reveal that the electrocatalytic anodic currents areobserved only in the presence of glucose oxidase and glucose, and thatno effect of the rotation speed of the external magnet on theelectrocatalytic anodic currents generated by ferrocene-functionalizedSiO₂ particles is observed.

Another experiment, as illustrated in FIGS. 4A and 4B, shows DNAanalysis using bioelectrocatalytic light emission. The Fe₃O₄ magneticparticles were silanized and a DNA primer (3) was covalently linked tothe silane thin film. The DNA functionalized magnetic particles werereacted with the DNA-analyte (4) resulting in a double stranded (ds) DNAhelix. The ds-DNA functionalized magnetic particles were reacted with adoxorubicin intercalator (5) that binds specifically to the ds-DNA. Thisintercalator is an electrochemically active quinone that can be reducedelectrochemically and can further reduce O₂, resulting in production ofhydrogen peroxide, H₂O₂. The electrocatalytically produced H₂O₂, in thepresence of horseradish peroxidase (HRP) and luminol, generates lightemission. The emitted light is an analytical signal reporting on thepresence of the intercalator and thus on the presence of the analyte DNA(4). The light emission intensity depends on the rate of theelectrocatalytic reduction of O₂. This rate is enhanced upon therotation of the modified magnetic particles, thus resulting in theamplification of the light emission.

In another experiment, DNA analysis was carried out usingbioelectrocatalytic precipitation of an insoluble material. The system,illustrated in FIG. 5, is similar to the one described above, but theelectrocatalytically generated H₂O₂ in the presence of HRP and4-chloronaphthol (6) results in the precipitation of the insolubleproduct (7). The insoluble product isolates the electrode surface. Thiseffect can be measured by means of Faradaic impedance orchronopotentiometry. The extent of the electrode isolation depends onthe rate of H₂O₂ production. This rate is enhanced by the rotation ofthe modified magnetic particles, thus resulting in the amplification ofthe signal.

A new immunosensor is illustrated in FIGS. 6A-C. Magnetic particles weresilanized with aminosilan as described above. An antigen that is acarboxylic derivative of dinitrophenyl, (8), is covalently coupled tothe amino groups of the siloxane layer at the surface of magneticparticles. The coupling reaction with the silanized magnetic particles,10 mg, proceeds with (8) at a concentration of 1 mM in the presence ofEDC, 5 mM, in 0.1 M HEPES buffer, pH 7.2, for 2 hours. Then the(8)-derivatized magnetic particles were washed with water in order toremove all unbound antigen molecules. The antigen modified magneticparticles are reacted with various concentrations of DNP-antibody, (9)(DNP being the abbreviation of dinitrophenol), (from 2 ng per mL to 50ng per mL) in 0.1 M phosphate buffer, pH 7.0, for 30 minutes. Then theantibody/antigen-functionalized magnetic particles are reacted withanti-DNP-antibody conjugated with the enzyme horseradish peroxidase(HRP), (10), 100 ng per mL, for 30 minutes. This secondaryanti-DNP-antibody, (10), is capable of binding to the primaryDNP-antibody, but not to the DNP-antigen (8). Thus, the amount of theHRP-conjugate-anti-DNP-antibody, (10), bound to the magnetic particlesis dependent on the presence of the DNP-antibody and it is proportionalto the later concentration. The described procedure of the magneticparticles functionalization with the antigen, (8), the DNP-antibody,(9), and the HRP-conjugate-anti-DNP-antibody, (10), is shown in FIG. 6A.The enzyme HRP uses hydrogen peroxide (H₂O₂) and luminol to producelight. Thus, H₂O₂ may be introduced to the sample as part of the assaysolution. However, in this example, another kind of functionalizedmagnetic particles is used to produce H₂O₂ and thus activate the systemelectrochemically, as illustrated in FIG. 6B. The silanized magneticparticles are reacted with 2,3-dichloro-1,4-naphthoquinone, (11), inethanolic suspension (5 mL) containing 10 mg of magnetic particles and100 mg of the quinone (11) for 3 minutes upon boiling the ethanolicsuspension. Then the quinone derivatized magnetic particles are washed 3times with ethanol and once with water. A system implementing thefunctionalized particles illustrated in both FIGS. 6A and 6B isillustrated in FIG. 6C. The system is composed of 10 mg of the quinone(11)-functionalized magnetic particles produced according to FIGS. 6Band 10 mg of the magnetic particles created according to FIG. 6A. Thesystem also includes an Au-plate electrode and the rotating magnet belowthe electrode. The solution also includes luminol, 1×10⁻⁵ M, in 0.1 Mphosphate buffer, pH 7.0, saturated with air. A light detector is fixedabove the solution. A potential of −0.6 V (vs. SCE) is applied to theelectrode, that provides electrochemical reduction of oxygen dissolvedin the solution. This reduction is catalysed by the quinone (11) andresults in the formation of hydrogen peroxide (H₂O₂). The hydrogenperoxide reacts with luminol in the presence of the enzyme HRP resultingin the light emission detected by a light detector.

FIGS. 7A and 7B show the results of the immunosensing system illustratedin FIG. 6C. FIG. 7A shows the intensities of the light emission withoutrotation of the magnetic particles (curve a) and with 100 rpm (rotationsper minute) (curve b). The signal is amplified because of the enhancedmass transport in the system upon the rotation (transport of oxygen tothe quinone, hydrogen peroxide from the quinone to the HRP, and luminolto the HRP). The light emission depends on the amount of the boundenzyme HRP, but its concentration is dependent on the concentration ofthe DNP-antibody (9) (analyte). FIG. 7B shows two calibration plots (thelight signal intensity vs. the DNP-antibody concentration): withoutrotation (a) and with 100 rpm (b). The ratio between the correspondingexperimental points on curves (b) and (a) presents the amplificationfactor achieved upon the rotation of the magnetic particles. It shouldbe noted that the amplification factor is dependent on the rotationspeed (however, the dependence is not linear—see the previous examples).

FIG. 8A illustrates an embodiment of the invention providing thedetection of a DNA analyte by use of DNA-functionalized magneticparticles, DNA labeled with biotin and an avidin-HRP.Amine-functionalized borosilicate-based magnetic particles (5 μm, MPG®Long Chain Alkylamine, CPG Inc.) were modified with a DNA primer (12)using the heterobifunctional crosslinker 3-maleimidopropionic acidN-hydroxysuccinimide ester. The coverage of the particles was estimatedusing the Oligreen® reagent (ssDNA Quantitation Assay Kit MolecularProbes, Inc.) to be ca. 52,000 oligonucleotide molecules-particle⁻¹. Theprimer (12) is complementary to a part of the target sequence (13). The(12)-functionalized magnetic particles are hybridized in a single stepwith a mixture that includes (variable concentrations) the target (13)and the biotin-labeled nucleic acid, (14), that is complementary to thefree segment (13). The three-component double-stranded DNA assembly(12)/(13)/(14) is then interacted with avidin-horseradish peroxidase(HRP) that acts as a biocatalytic label.

According to one embodiment, depicted in FIG. 8B the DNA/avidin-HRPfunctionalized magnetite particles of FIG. 8A are subsequently mixedwith magnetite particles modified with the naphthoquinone unit (15). Themixture of the magnetic particles is then attracted to an electrodesupport by means of an external magnet. Electrochemical reduction of thenaphthoquinone to the respective hydroquinone results in the catalyzedreduction of O₂ to H₂O₂. The electrogenerated H₂O₂ leads, in thepresence of luminol, (16), and the HRP enzyme label to the generation ofthe chemiluminescence signal.

The avidin-HRP approaches the electrode only if the target DNAhybridizes with the magnetic particles, provided that non-specificadsorption does not take place. Thus, chemiluminescence occurs only ifthe target DNA (13), is in the analyzed sample. Furthermore, the lightintensity relates directly to the number of recognition pairs of (12)and (13) associated with the electrode, and thus it provides aquantitative measure to the concentration of (13) in the sample.

The rotation of the particles on the barrier surface by means of therotating external magnet results in the enhanced electrogeneratedchemiluminescence, since the magnetic particles behave as rotatingmicroelectrodes, where the interaction of O₂ and luminol with thecatalysts on the electrode is controlled by convection rather than bydiffusion. Thus, the rotation of the magnetic particles is anticipatedto yield the amplified detection of DNA.

It should be appreciated that electrogeneration of H₂O₂ is not anecessary part of the invention, and according to a differentembodiment, H₂O₂ may be directly introduced to the assay sample. In suchcase, the electrode is also not necessary. However, in such alternativeembodiment, as H₂O₂ is not localized near an electrode, excessavidin-HRP must be removed from the assayed sample prior to providingthe reaction conditions.

In an experiment carried out essentially according to FIGS. 8A and 8B, asample of (12)-functionalized magnetic particle was interacted with(13), 1.4×10⁻⁸ M, in the presence of the biotinylated nucleic acid,(14), 2×10⁷ M. The resulting double-stranded (12)/(13)/(14)tri-component system was collected by the external magnet, washed with0.2 M phosphate buffer (pH 7.4), and then reacted with the avidin-HRPconjugate and again collected by the external magnet. The resultingparticles were suspended in the electrochemical cell together with thenaphthoquinone (15)-modified magnetic particles, 2 mg·ml⁻¹. In FIG. 9A,curve (a) shows the emitted light intensity upon the collection of themagnetic particles on the electrode by means of the external magnet, andthe application of a potential step on the electrode from 0 V to −0.5 Vand back. FIG. 9A, curve (b)-(d) shows the emitted light intensitiesupon the rotation of the particles by means of the external magnet,using different rotation speeds. Increase of the rotation speed enhancesthe intensity of the emitted light, and the resulting light intensityrelates linearly to ω^(1/2) (ω=rotation speed), as expected forelectrocatalytic rotating microelectrodes. In a control experiment thatlacks (13) in the hybridization step, no light emission is detected,indicating that no non-specific adsorption of (14) or the avidin-HRPconjugate takes place. The light intensity emitted from the systemrelates to the surface coverage of the avidin-HRP conjugate, and this iscontrolled by the amount of (13)/(14) associated with the particles andthus determined by the concentration of (13). FIG. 9B, shows the derivedcalibration curves corresponding to the emitted light intensities uponanalyzing different concentrations of (13) and recorded at differentrotation speeds. FIG. 9A, curve (e), shows the light intensity observedupon the analysis of the mutant (13a), that includes a 7-base mutationsequence in respect to (13), 1×10⁻⁷ M, at a rotation speed of 2000r.p.m. according to the embodiment of FIGS. 8A and 8B. No emitted lightdue to non-specific adsorption of the avidin-HRP conjugate on thesurface, is observed. This light intensity is considered as thebackground signal, and thus (13) can be sensed in this example with adetection limit of 1×10⁻¹⁴ M at ω=2000 r.p.m. (S/N>3).

The following examples show use of the detection of a DNA analyteaccording to this invention where the reaction signal is amplified usingpolymerase chain reaction. In those examples, the following experimentalconditions and materials were used:

-   -   Amine-functionalized borosilicate-based magnetic particles (5        μm, MPG® Long Chain Alkylamine, CPG Inc.), Biotin-21-dUTP        (Clontech). The heterobifunctional crosslinker        3-maleimidopropionic acid N-hydroxysuccinimide ester,        oligonucleotides (17), (18), (19) and (20), Avidin-HRP        conjugate, dNTP's, Biotin-11-dCTP, Taq Polymerase, 10×PCR buffer        and all other compounds were purchased from Sigrna and used as        received.    -   Preparation of DNA-functionalized magnetic particles: 30 mg of        the amino-functionalized magnetic particles (MPG® Long Chain        Alkylamine, CPG Inc.) were activated by reaction with the        heterobifunctional crosslinker 3-maleimidopropionic acid        N-hydroxysuccinimide ester (10 mg, Sigma) in 1 ml of DMSO. After        4 hrs of incubation at room temperature, the particles were        collected with and external magnet and thoroughly washed with        DMSO and water. The maleimido-activated particles were then        reacted with 20-30 O.D. of the thiolated oligonucleotide in        phosphate buffer 0.1M, pH 7.4 for a period of 8 hrs. (The        thiolated nucleotide was freshly reduced with DTT and separated        on a Sephadex G-25 column prior to the reaction with the        functionalized particles). Finally, the magnetic particles were        washed with water and phosphate buffer 0.1M, pH7.4. In order to        keep the DNA-modified particles for periods longer than one        week, 1% w/v sodium azide was added, and the particles were kept        at 4° C. The oligonucleotide content on the magnetic particles,        before and after enzymatic DNase treatment (10 units DNase, 30        min at 37° C.) was measured by the use of the Oligreen® reagent        (ssDNA Quantitation Assay Kit Molecular Probes, Inc.).    -   φ: (a) For single-point-mutation detection: denaturation 30 sec,        94° C.; annealing 30 sec, 55° C.; polymerization 5 sec,        72° C. (b) For Viral detection: denaturation 30 sec, 94° C.;        annealing 30 sec, 55° C.; polymerization 15 sec, 72° C.    -   An Au-coated (50 nm gold layer) glass plate (Analytical-μSystem,        Germany) was used as a working electrode (0.3 cm² area exposed        to the solution). An auxiliary Pt electrode and a        quasi-reference Ag electrode were made from wires of 0.5 mm        diameter and added to the cell. The quasi-reference electrode        was calibrated vs. saturated calomel electrode and the        potentials are given vs. SCE. An open electrochemical cell (230        μL) that includes the Au-electrode in a horizontal position and        a light detector linked to a fiber optics enabled easy light        emission measurements upon application of the appropriate        potential to the modified working electrode. The electrochemical        measurements were performed using a potentiostat (EG&G,        model 283) connected to a computer (EG&G Software 270/250 for).        All the measurements were performed in 0.01 M phosphate buffer        solution, pH 7.0, at room temperature. The        electrochemically-induced chemiluminescence was measured with a        light detector (Laserstat, Ophir) linked to an oscilloscope        (Tektronix TDS 220). The light detector was connected to the        electrochemical cell by an optical fiber. The background        electrolyte solution was equilibrated with air and included        luminol, 1×10⁻⁶ M.

FIG. 10 depicts a method for the amplified detection of the viral MP13φDNA, using functionalized magnetic particles. Magnetic particles (MPG®Long Chain Alkylamine, 5 μm diameter, CPG Inc.) are modified with thethiolated primer (17) using the heterobifunctional cross-linker3-maleimidopropionic acid N-hydroxysuccinimide ester, as outlined inFIG. 11. The average coverage of the magnetite particles was determinedby using the Oligreen® reagent (Molecular Probes, Inc.) and correspondsto ca. 50,000 oligonucleotide units-particle⁻¹. The number of nucleicacids that are associated with the particles and accessible to anexternal enzyme was estimated by subjecting the (17)-functionalizedmagnetic particles to DNase and by the subsequent determination of thecontent of the DNA that is cleaved off. It was found that ca. 20,000oligo units-particle⁻¹ are cleaved off, implying that only ca. 40% ofthe particle-linked nucleic acids are accessible to the enzyme. As shownin FIG. 10, The (17)-modified magnetic particles are hybridized with theMP13φ DNA (7229 bases) and are subjected to polymerization in thepresence of a mixture of dGTP; dATP; dCTP and biotinylated dUTP(b-dUTP). The polymerization introduces into the replica a high numberof biotin labels. The replication is followed by thermal cycles thatresult in the dissociation of the analyzed MP13φ DNA, itsre-hybridization with other oligonucleotide primers associated with themagnetic particles, and the subsequent polymerization and formation ofnew replica containing a high number of biotin label units. Bycontrolling the number of thermal cycles, the replication on theparticles' surface yields very high densities of biotin-labeled nucleicacids on the magnetic particles. The thermal cycles were conducted for30 sec each, which translate to a replication efficiency of ca.500-bases per cycle. This relatively low replication efficiency waspurposely designed in order to eliminate steric crowding by the nucleicacid replica on the magnetite particles that might perturb the cycliclabeling of the particle by the biotin labels. The resultingbiotin-labeled magnetic particles are then separated by means of theexternal magnet, reacted with avidin-horseradish peroxidase (HRP), andagain separated by the external magnet and washed with a phosphatebuffer solution. A mixture of the avidin-functionalized magneticparticles, 1 mg, and naphthoquinone-modified magnetic particles (15)(not shown) is then introduced into the electrochemical cell. Themagnetic particles are then attracted to the electrode by means of theexternal magnet. Upon the application of a potential on the electrodethat reduces the naphthoquinone to the respective hydroquinone, theelectrocatalyzed reduction of O₂ to H₂O₂ occurs. The HRP-catalyzedoxidation of luminol, by the electrogenerated H₂O₂ results inchemiluminescence and the emission of light.

FIG. 12, curve (a), shows the emitted light intensity upon analyzingMP13 φDNA, 8.3×10⁻⁹M, according to the above, and by the application ofa potential step on the electrode from 0 V to −0.5 V and back (the E°for the naphthoquinone-modified particles at pH=7 is 0.4 V). In acontrol experiment in which all the analysis steps were applied on asample that lacks MP13φ? DNA, (curve (e)) no light emission wasobserved, indicating that no non-specific adsorption of the avidin-HRPconjugate on the electrode, or on the magnetic particles, takes place,FIG. 12.

The effect of rotation of the magnetic particles by means of therotating external magnet is depicted in FIG. 12, curves (b)-(d). As therotation speed of the magnetic particles is elevated, the emitted lightintensity increases, and a linear relationship between the intensity ofemitted light and ω^(1/2) (ω=the rotation speed) is observed, FIG. 12(inset).

At a constant rotation speed of the particles, the intensity of emittedlight is controlled by the surface coverage of the labeled nucleic acidassociated with the magnetic particles, and this relates to theconcentration of MP13φ? DNA in the analyzed sample during thereplication cycles. FIG. 13 shows the emitted light intensity upon theanalysis of different concentrations of MP13φ? DNA at a rotation speedof ω=2000 r.p.m.

A further example of the invention employs functional magnetic particlesfor the amplified detection of single base mismatches in DNA. This isexemplified by the analysis of the mutant sequence (18), where a G-baseexchanges the A-base in the normal sequence gene (19), as shown in FIG.14. The magnetic particles are functionalized with the nucleic acid (20)that is complementary to the mutant sequence, (18), and the normal genesequence (19), up to one base prior to the mutation site. Interaction ofthe (20)-modified magnetic particles with the samples that includeeither (18) or (19) results in their hybridization with the particles.Treatment of the hybridized assemblies associated with the magneticparticles with polymerase and biotinylated-dCTP, followed by theapplication of thermal dissociation/annealing/labeling cycles results inthe multi-labeling of the magnetic particles with biotin units upon theanalysis of (18), whereas no biotin labels are introduced upon theanalysis of (19). The subsequent interaction of the particles with theavidin-HRP conjugate, followed by the separation of the particles bymeans of the external magnet yield the biocatalytically labeledparticles. Mixing of the resulting particles with thenaphthoquinone-modified particles (15)(not shown) in the electrochemicalcell, followed by their attraction to the electrode by means of theexternal magnet leads to the electrocatalyzed reduction of O₂ to H₂O₂,and in the presence of luminol, to the emission of light upon theanalysis of (18), while no light is detected upon the analysis of (19).Rotation of the magnetic particles by means of an external magnet isthen expected to amplify the emitted light since the electrogeneratedchemiluminescence is controlled by convection of the respectivesubstrates to the particles.

FIG. 15 shows the emitted light intensity at a rotation speed of 2000r.p.m., upon analyzing (18), 1×10⁻⁹M, curve (a), and (19), 1.4×10⁻⁶M,curve (d), all according to FIG. 14. A potential step from 0 V to −0.5 Vand back is applied on the electrode in order to activate theelectrocatalyzed reduction of O₂, and to drive the secondarychemiluminescent process. No light emission is observed upon theanalysis of (19), indicating that no biotin labels were incorporatedinto the nucleic acid-modified magnetic particles. Clearly, lightemission is observed only upon the analysis of the mutant sequence. FIG.15, curves (a)-(c), shows the light emitted from the system upon therotation of the magnetic particles at different rotation speeds. FIG. 16shows the light emitted upon analyzing different concentrations of (18),at different rotation speeds. The intensity of the emitted lightincreases as the rotation speed of the particles is elevated, (P∝ω^(1/2)), implying that the processes at the electrode support arecontrolled by convection. The mutant sequence (18) is analyzed with adetection limit of 1×10⁻¹⁷ M. The concentration of (19) in the analyzedsample is 10³-fold higher than that of (18), and still no light emissionis observed upon analyzing (19). This implies that no non-specificassociation of the HRP conjugate to the magnetic particles occurs.

In conclusion, this example described a magnetically amplified DNAanalysis process. Several consecutive steps in the process lead to theoverall amplification: (i) The thermal cyclic replication of the analyteon the magnetic particles leads to the incorporation of a high number oflabel-units into the nucleic acids linked to the particles. (ii) Theelectrocatalytic generation of O₂ at the electrode, and the coupledbiocatalyzed light emission yield numerous product molecules or photonsas a result of a single recognition event. (iii) The rotation of themagnetic particles leads to the amplified light emission since thetransport of the substrates for the electrocatalytic and biocatalyticprocesses at the particles are convection-controlled. Using thesemethods, very high sensitivities were achieved.

Yet another example of the invention is the detection of an enzyme in agiven sample. Such detection of the enzyme analyte telomerase isschematically depicted in FIG. 17A. The analyte, telomerase, is aribonucleoprotein complex capable of synthesizing new telomers by theaddition of telomeric repeats to the 3′-end of chromosomal DNA.Accordingly, the recognition agent in this example is a nucleic acidsequence (21) that comprises a 6 T-base linker unit followed by acharacteristic sequence recognized by the telomerase.Amine-functionalized magnetic particles (5 μm diameter) were activatedwith the bifunctional reagent 3-maleimidopropionic acid-N—hydroxysuccinimide ester, substantially as outlined in FIG. 11 (thistime with sequence (21) instead of sequence (17)). Themercaptohexyl-modified nucleic acid, (21), was covalently linked to themagnetic particles. Specifically, 30 mg of the amino-functionalizedmagnetic particles (MPG® Long Chain Alkylamine, CPG Inc.) were activatedby reaction with the heterobifunctional crosslinker 3-maleimidopropionicacid N-hydroxysuccinimide ester (10 mg, Sigma) in 1 mL of DMSO. After 4hrs of incubation at room temperature, the particles were collected withan external magnet and thoroughly washed with DMSO and water. Themaleimido-activated particles were then reacted with 20-30 O.D. of thethiolated oligonucleotide in phosphate buffer 0.1 M, pH 7.4 for a periodof 8 hrs. (The thiolated nucleotide was freshly reduced with DTT andseparated on a Sephadex G-25 column prior to the reaction with thefunctionalized particles). Finally, the magnetic particles were washedwith water and phosphate buffer, 0.1 M, pH 7.4. In order to keep theDNA-modified particles for periods longer than one week, 1% w/v sodiumazide was added, and the particles were kept at 4° C. Theoligonucleotide content on the magnetic particles, before and afterenzymatic DNase treatment (10 units DNase, 30 min at 37° C.) wasmeasured by the use of the Oligreen® reagent (ssDNA Quantitation AssayKit Molecular Probes, Inc.)

As schematically shown in FIG. 17A, the functional magnetic particlescomprising the recognition agent (21) are treated with cell extract(which is assayed for the presence of the analyte) in the presence of amixture of nucleotides dNTP that includes biotin labeled dUTP. Theassociation of telomerase to the recognition agent is followed bytelomerization that involves the labeling of the newly synthesizedchains with biotin integrated into the telomeric repeats. The subsequentbinding of avidin-horseradish peroxidase (HRP) introduces thebiocatalytic labels into the telomer chains. The magnetic particles arecollected by attraction to the bottom of the analyzing flask by means ofan external magnet and washed to remove any residual cell extract ornon-specifically absorbed HRP conjugate.

As schematically shown in FIG. 17B the resulting particles of FIG. 17Aare then mixed with the naphthoquinone-functionalized magnetiteparticles (15) synthesized by the reaction of 3,4-dichloronaphthoquinonewith aminoethylamine-modified magnetic particles. The mixture ofparticles are introduced into an electrochemical cell that includesluminol, (16). Upon the application of a potential step that reduces thequinone to hydroquinone, the electrocatalyzed reduction of O₂ to H₂O₂proceeds. The resulting H₂O₂ mediates the HRP catalyzed oxidation ofluminol with the concomitant emission of light.

The electrogenerated luminescence is observed only if the HRP labelsbind to the telomerase units, and this occurs only provided telomerase(the analyte) exists in the analyzed cell extract. Also, the intensityof electrogenerated luminescence is controlled by the content oflabels/avidin-HRP conjugates associated with the particles, and this isdetermined by the amount of telomerase enzyme in the sample.Furthermore, the rotation of the magnetic particles by means of theexternal rotating magnet further amplifies the emitted light intensity.Upon rotation of the particles, the electrocatalyzed reduction of O₂ andthe interaction of H₂O₂ with luminol are controlled by convection ratherthan by diffusion, leading to enhanced (amplified) light emission.

FIG. 18A depicts the analysis of 293-kidney cancer cells extractaccording to FIGS. 17A and B. It shows the light emitted from the systemupon the analysis of a 100,000 cells extract dilution, while applying apotential step from 0 V to −0.5 V and rotating the particles atdifferent speeds. The intensity of light emitted from the system isenhanced as the rotation speed increases. Provided that the functionalmagnetic particles behave in the analytical system as rotatingmicroelectrodes, and that the electrocatalyzed generation of light iscontrolled by convection of the substrates to the rotating electrode, alinear dependency between the emitted light and ω^(1/2) (ω=rotationspeed, rad·s⁻¹) should exist. FIG. 18B shows that indeed a linearrelation between the electrogenerated light and ω^(1/2) exists. Incontrol experiments where the naphthoquinone-functionalized magneticparticles or the avidin-HRP conjugate are excluded from the system, nolight emission from the systems is detected at any rotation speed of theparticles. These experiments confirm that the electrogeneratedchemiluminescence originates from the primary electrocatalyzed reductionof O₂ to H₂O₂ and the subsequent HRP-mediated oxidation of luminol byH₂O₂.

The electrogenerated chemiluminescence at constant rotation speed iscontrolled by the number of the cancer cells in the extract. FIG. 19shows the light emitted from the electrochemical cell upon the analysisof different concentrations of the 293-kidney cancer cells at constantrotation speeds that corresponds to (i) 0 rpm, (ii) 60 rpm and (iii)2000 rpm, and according to the detection route depicted in FIGS. 17A andB. In these systems, a potential step from 0.0 V to −0.5 V is applied onthe functional particles. FIG. 19 inset, shows the light emitted fromextracts that include 100 and 10 293-kidney cancer cells, respectively.For comparison, no generation of light is observed upon the applicationof the entire analysis protocol in the absence of cells or in thepresence of 293-kidney cancer cells extract heated to 90° C. prior tothe analysis process in order to inactivate the telomerase activity.This implies that no non-specific binding of the avidin-HRP conjugate tothe magnetic particles or the electrode takes place.

Similar results are observed upon the analysis of telomerase in culturedHeLa cells. FIG. 20A shows the electrogenerated chemiluminescence uponanalysis of a HeLa cells extract, according to FIG. 17, and usingdifferent rotation speeds of the magnetic particles. The intensity ofemitted light is enhanced upon increasing the rotation speed of theparticles, and a linear relation between the intensity of generatedlight and ω^(1/2) (rad·s⁻¹) is observed, as showed in FIG. 20A, inset.The intensities of electrogenerated chemiluminescence upon the analysisof different concentrations of HeLa cells extracts are shown in FIG.20B. In this experiment, the magnetic particles are rotated at aconstant rotation speed of (i) 0 rpm, (ii) 60 rpm or (iii) 2000 rpm.Clearly, the light emitted upon the analysis of 10 HeLa cells is easilydetectable.

FIG. 21 shows the electrogenerated chemiluminescence upon analysis oftelomerase activity HeLa cells extract, curve (a), a 293-kidney cancercells extract, curve (b), and the analysis of a NHF (Normal Humanfibroblast) cells extract, curve (c). Clearly, the electrogeneratedchemiluminescence observed upon analyzing a 100-fold higher content ofnormal cells is ca. 200-fold lower than the light emitted from 1000 293kidney cancer cells. The minute light emitted from the system thatincludes the normal NHF cells may be attributed to the non-specificadsorption of residual quantities of the avidin-HRP conjugate to themagnetic particles. The light generated in the system that includes theNHF cells may be considered as the background light level of theanalysis scheme.

In addition, the capability to diagnose cancer in a suspected tissue isexemplified in FIG. 22. Different tissues from patients with lung cancerwere assayed. The electrogenerated chemiluminescence intensitiesobtained upon analyzing healthy cells, adenocarcinoma and squamousepithelial carcinoma cells are shown in FIG. 22 and compared to thelight signals observed upon analyzing healthy tissue or normal cellextracts. The chemiluminescence signals (telomerase activity) obtainedfrom the carcinoma tissues were significantly higher than the minutechemiluminescence signal obtained from healthy tissue cells extracts.

This example clearly shows that the invention may be useful for thedetection of telomerase as a rapid method to identify cancer cells andto monitor anti-cancer therapeutic treatments.

In all of the above telomerase assays, an Au-coated (50 nm gold layer)glass plate (Analytical-μSystem, Germany) was used as a workingelectrode (0.3 cm² area exposed to the solution). An auxiliary Ptelectrode and a quasi-reference Ag electrode were made from wires of 0.5mm diameter and added to the cell. The quasi-reference electrode wascalibrated vs. saturated calomel electrode, and the potentials are givenvs. SCE. An open electrochemical cell (230 μL) that includes theAu-electrode in a horizontal position and a light detection linked to afiber optics, enabled easy light emission measurements upon applicationof the appropriate potential to the modified working electrode. Theelectrochemical measurements were performed using a potentiostat (EG&G,model 283) connected to a computer (EG&G Software 270/250 for). All themeasurements were performed in 0.01 M phosphate buffer solution, pH 7.0,at room temperature. The electrochemically-induced chemiluminescence wasmeasured with a light detection (Laserstat, Ophir) linked to anoscilloscope (Tektronix TDS 220). The light detector was connected tothe electrochemical cell by an optical fiber. The background electrolytesolution was equilibrated with air and included luminol, 1×10⁻⁶ M.

1. A method for determining an analyte in an assayed sample, comprising:(i) providing magnetic particles carrying a recognition agent that bindsto or reacts with the analyte, such that, under assay conditions, saidbinding or reaction gives rise to a reaction that yields a reactionsignal; (ii) contacting said magnetic particles with the assayed sample,drawing the magnetic particles to a barrier surface through a magnetproximal to the barrier surface, providing the assay conditions andinducing the magnetic particles to rotate or vibrate in response to anexternal magnetic field that changes in time in a periodical manner,giving rise to a reaction signal that is enhanced during the rotation orvibration; and (iii) reading said reaction signal.
 2. The methodaccording to claim 1, wherein said magnetic field is a rotating magneticfield.
 3. The method according to claim 2, wherein said rotatingmagnetic field is induced by a rotating magnet.
 4. The method accordingto claim 1, wherein said magnetic field is a vibrating magnetic field.5. The method according to claim 1, wherein said reaction is a redoxreaction.
 6. The method according to claim 1, wherein said magneticparticles are confined to a support.
 7. The method according to claim 1,wherein the recognition agent and the analyte react with one another ina manner to yield a reaction product.
 8. The method according to claim1, wherein the recognition agent is a catalyst that can induce areaction in which the analyte is converted into a product.
 9. The methodaccording to claim 1, wherein the analyte and the recognition agent forma recognition pair and the detection of the analyte is based on the useof a reagent that binds to the formed pair.
 10. The method according toclaim 9, wherein the analyte is a protein analyte and the reagent is anantibody capable of binding to said analyte.
 11. The method according toclaim 9, wherein said analyte is a DNA analyte.
 12. The method accordingto claim 11, wherein the assay conditions comprise a DNA polymerase andnucleotide bases, at least one of said nucleotide bases being bound to adetectable moiety.
 13. The method according to claim 12, wherein saiddetectable moiety is biotin and the assay conditions further comprise anavidin bound enzyme.
 14. The method according to claim 13, wherein theenzyme is horseradish peroxidase and the reaction signal is lightemission.
 15. The method according to claim 12, wherein the DNApolymerase is Taq Polymerase and the reaction conditions are such thatenable polymerase chain reaction to take place.
 16. The method accordingto claim 11, allowing the detection of at least one base mismatch. 17.The method according to claim 7, wherein the analyte is a catalyst thatcan induce a reaction in which the recognition agent is converted into aproduct.
 18. The method according to claim 7, wherein the recognitionagent comprises a catalyst that can induce a reaction in which theanalyte is converted into a product.
 19. The method according to claim17, wherein the catalyst is an enzyme.
 20. The method according to claim19, wherein the enzyme is telomerase.
 21. The method according to claim20, wherein the assayed sample comprises cellular extract.
 22. Themethod according to claim 1, wherein the analyte and the recognitionagent form a recognition pair and the detection of the analyte is basedon the use of a reagent that binds specifically to the analyte, wheresaid analyte is first bound to the recognition agent.
 23. The methodaccording to claim 10, wherein said analyte is an antibody analyte. 24.The method according to claim 1, wherein at least one of the componentsof the chemical system remain during the analysis dissolved in themedium of the assayed sample.
 25. The method according to claims 1,wherein the reaction signal is selected from electrical signal, lightemission signal, calorimetric signal and formation of a precipitate. 26.A system for determining an analyte in an assayed sample, the systemcomprising: (i) a cell with a barrier surface (ii) a sub-system forcausing the magnetic particles to rotate or vibrate, said subsystemcomprising a motor associated with the magnet that causes the magneticparticles to rotate or vibrate; (iii) magnetic particles havingimmobilized thereon a recognition agent such that in the presence of theanalyte, a reaction occurs yielding a reaction signal, said signal beingenhanced during the rotation or vibration of said magnet; (iv) sensingmember for sensing said reaction signal; and (v) reader for reading saidreaction signal.
 27. The system according to claim 26, wherein saidreaction is a redox reaction and said sensing member is an electrode.28. The system according to claim 27, wherein said recognition agentcomprises at least one molecule capable to transfer electrons betweensaid electrode and said analyte.
 29. The system according to claim 26,wherein the recognition agent and the analyte react with one another ina manner to yield a reaction product.
 30. The system according to claim26, wherein the analyte is a catalyst that can induce a reaction inwhich the recognition agent is converted into a product.
 31. The systemaccording to claim 26, wherein the recognition agent comprises acatalyst that can induce a reaction in which the analyte is convertedinto a product.
 32. The system according to claim 26, wherein theanalyte and the recognition agent form a recognition pair and thedetection of the analyte is based on the use of a reagent that binds tothe formed pair.
 33. The system according to claim 26, wherein saidanalyte is a DNA analyte.
 34. The system according to claim 26, whereinthe analyte and the recognition agent form a recognition pair and thedetection of the analyte is based on the use of a reagent that bindsspecifically to the analyte, where said analyte is first bound to therecognition agent.
 35. The system according to claim 34, wherein saidanalyte is an antibody analyte.
 36. The system according to claim 26,wherein said signal is selected from electrical signal, light emissionsignal, calorimetric signal and formation of a precipitate.
 37. Themethod according to claim 20 for the detection of cancer cells.
 38. Themethod according to claim 37 for the detection of cancer cellscomprising: (i) providing magnetic particles carrying a DNA recognitionagent that serves as a primer for telomerase, such that, under assayconditions, the telomerase reaction enables a reaction that yields areaction signal; (ii) providing an assay sample comprising cellularextract from one or more cells suspected of being cancerous; (iii)contacting said magnetic particles with the assayed sample, drawing themagnetic particles to a barrier surface through a magnet proximal to thebarrier surface, providing the assay conditions and inducing themagnetic particles to rotate or vibrate, giving rise to a reactionsignal that is enhanced during the rotation or vibration; (iv) readingsaid reaction signal; and (v) comparing said reading with a readingobtained from a control assay sample not containing cancerous cells, ahigher reading in the assay sample than in the control assay sampleindicating that said suspected cells are cancerous.
 39. The methodaccording to claim 38, wherein said reaction signal is light emission.40. A method according to claim 1 for the detection of more than oneanalyte comprising: (i) providing magnetic particles carrying more thanone recognition agent, each of which recognition agents binds to orreacts with at least one of said analytes, such that, under assayconditions, each binding or reaction gives rise to a reaction thatyields a distinguishable reaction signal, and in the presence of morethan one of said analytes more than one distinguishable reaction signalsare yielded; (ii) contacting said magnetic particles with the assayedsample, drawing the magnetic particles to a barrier surface through amagnet proximal to the barrier surface, providing the assay conditionsand inducing the magnetic particles to rotate or vibrate in response toan external magnetic field that changes in time in a periodical manner,giving rise to said distinguishable reaction signals; and (iii) readingsaid distinguishable reaction signals.
 41. The method of claim 40,wherein step (ii) comprises reading the distinguishable reaction signalsusing different reading means.
 42. The method of claim 40, wherein steps(ii) and (iii) are repeated more than once, using different assayconditions.
 43. A system for determining more than one analyte in anassayed sample, the system comprising: (i) a cell with a barrier surface(ii) a sub-system for causing the magnetic particles to rotate orvibrate; (iii) magnetic particles having immobilized thereon more thanone recognition agent such that in the presence of the analytes,reactions occur yielding distinguishable reaction signals, said signalsbeing enhanced during the rotation or vibration of said magnet; (iv)more than one sensing members for sensing each of said distinguishablereaction signals; and (v) one or more readers for reading said reactionsignal.
 44. A system for determining an analyte in an assayed sample,the system comprising: (i) a cell with a barrier surface (ii) asub-system for causing the magnetic particles to rotate or vibrate, saidsubsystem comprising a motor associated with the magnet that causes themagnetic particles to rotate or vibrate; (iii) magnetic particles havingimmobilized thereon a recognition agent such that in the presence of theanalyte, a reaction occurs yielding a reaction signal, said signal beingenhanced during the rotation or vibration of said magnet; and (iv)sensing member for sensing said reaction signal, whereby the signal isindicative of the presence and/or amount of said analyte in the sample.45. A system for determining more than one analyte in an assayed sample,the system comprising: (i) a cell with a barrier surface (ii) asub-system for causing the magnetic particles to rotate or vibrate;(iii) magnetic particles having immobilized thereon more than onerecognition agent such that in the presence of the analytes, reactionsoccur yielding distinguishable reaction signals, said signals beingenhanced during the rotation or vibration of said magnet; and (iv) morethan one sensing members for sensing each of said distinguishablereaction signals; whereby each of said signals is indicative of thepresence and/or amount of an analyte in the sample.